Design, synthesis, biological evaluation and in silico studies of pyrazole‐based nh2‐acyl oseltamivir analogues as potent neuraminidase inhibitors

Article abstract of DOI:10.3390/ph14040371

Oseltamivir represents one of the most successful neuraminidase (NA) inhibitors in the current anti‐influenza therapy. The 150‐cavity of NA was identified as an additional binding pocket, and novel NA inhibitors have been designed to occupy the 150‐cavity

Full text of DOI:10.3390/ph14040371

pharmaceuticals
Article
Design, Synthesis, Biological Evaluation and In Silico Studies
of Pyrazole-Based NH2-Acyl Oseltamivir Analogues as Potent
Neuraminidase Inhibitors
Jiqing Ye 1 , Lin Lin 2, Jinyi Xu 3, Paul Kay-sheung Chan 2,4, Xiao Yang 2,* and Cong Ma 1,
*
1
State Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biology and
Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China;
jiqing.ye@connect.polyu.hk
2
3
4
Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital,
Shatin, Hong Kong, China; linlin@cuhk.edu.hk (L.L.); paulkschan@cuhk.edu.hk (P.K.-s.C.)
State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry,
China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China; jinyixu@cpu.edu.cn
Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong,
Shatin, Hong Kong, China
*
Correspondence: xiaoyang@cuhk.edu.hk (X.Y.); cong.ma@polyu.edu.hk (C.M.)
Abstract: Oseltamivir represents one of the most successful neuraminidase (NA) inhibitors in the cur-
rent anti-influenza therapy. The 150-cavity of NA was identified as an additional binding pocket, and
novel NA inhibitors have been designed to occupy the 150-cavity based on the structure informa-
tion of oseltamivir carboxylate (OC) in complex with NA. In this study, a series of C-5-NH2-acyl
derivatives of OC containing the pyrazole moiety were synthesized. Several derivatives exhibited
substantial inhibitory activity against NA. Moreover, in silico ADME evaluation indicated that the
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Ye, J.; Lin, L.; Xu, J.; Chan,
P.K.-s.; Yang, X.; Ma, C. Design,
Synthesis, Biological Evaluation and
In Silico Studies of Pyrazole-Based
NH2-Acyl Oseltamivir Analogues as
Potent Neuraminidase Inhibitors.
Pharmaceuticals 2021, 14, 371.
derivatives were drug-like with higher oral absorption rates and greater cell permeability than OC
.
Additionally, molecular docking studies revealed that the derivatives interacted with both the NA
enzyme active site and 150-cavity as expected. The results provided useful information for further
structural optimization of OC.
Keywords: influenza virus; neuraminidase inhibitor; 150-cavity; oseltamivir derivatives; pyrazole
https://doi.org/10.3390/ph14040371
Academic Editor: Daniela Catarzi
1. Introduction
Received: 24 March 2021
Accepted: 15 April 2021
Published: 16 April 2021
Seasonal flu epidemics and pandemics caused substantial mortality and economic
loss. Reports showed that up to 0.65 million respiratory deaths per annum are attributed
to seasonal flu globally [
(NP) and matrix protein (M1), influenza viruses are classified as types A, B, and C [
Different subtypes of influenza A virus were named according to two important surface
antigenic glycoproteins, hemagglutinin (HA) and neuraminidase (NA) [ ]. So far, 18 HA
subtypes (H1–18) and 11 NA subtypes (N1–N11) were identified [ ], in which several
different subtypes caused severe pandemics in the last century, including the 1918 Spanish
1
]. According to the antigenic differences in their nucleoprotein
2
].
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
3
4
flu (H1N1), 1957 Asian flu (H2N2), 1968 Hong Kong flu (H3N2) [
(H1N1 pdm09) [6].
5], and 2009 swine flu
Presently, there are three classes of anti-influenza drugs, consisting of the M2 chan-
nel inhibitors, the neuraminidase inhibitors (NAIs) [ ], and the recently approved cap-
dependent endonuclease inhibitor baloxavir marboxil [ ] (Figure 1), although the viral
resistance to all the drugs has been reported [ ]. NAIs have been approved as antivirals in
clinical use for about 20 years, and they have a mode of action of disrupting the release of
nascent viruses from host cells. Amongst all the NAIs, oseltamivir phosphate (OP, ethyl
ester prodrug) has become a first-line therapy since its approval in 1999 [10].
Copyright:
© 2021 by the authors.
7
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
8
9
Pharmaceuticals 2021, 14, 371. https://doi.org/10.3390/ph14040371
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 371
2 of 20
Figure 1. Representative anti-influenza drugs. M2 channel inhibitors: (
cap-dependent endonuclease inhibitor (vii).
i
,
ii); NAIs: (iii
–
vi) and
Phylogenetically, NAs can be divided into two groups: group 1 contains N1, N4,
N5, and N8, and group 2 contains N2, N3, N6, N7, and N9 [11]. Based on the structural
observations, Russel et al. disclosed that group-1 neuraminidases contain a so-called
150-cavity adjacent to their active sites, which suggests a new binding pocket for drug
design [12]. Besides, it was also reported that N2 NA could be induced to an open position
by the binding of oseltamivir carboxylate to NA [13]. Crystal structures of OC in complex
with NAs indicated that the C-5-NH2 group of OC close to the 150-cavity of NA might be
a possible modification site. On this basis, several C-5-NH2 modified OC derivatives have
been reported as 150-cavity binders, including second amine derivatives (viii–xiii) [14–17].
These derivatives inhibited NA at the nM level, and most of them possessed the selectivity
against group 1 NAs. Other derivatives, such as triazolylated analogue (xiv) [18], pyridyl
analogue (xv) [19], and C-5-NH2-acyl analogue (xvi) (Figure 2) [20], showed moderate
activity against NA.
Figure 2. Representative C-5-NH2 modified OC derivatives designed as 150-cavity binders: secondary amine derivatives
(viii–xiii), triazolylated analogue (xiv), pyridyl analogue (xv) and acyl analogue (xvi).

Pharmaceuticals 2021, 14, 371
3 of 20
Previously, we designed and synthesized a class of heterocycle-bearing secondary
amine derivatives of OC for probing the 150-cavity [17]. Results indicated that the introduc-
tion of heterocyclic side chains could maintain the antiviral activity of OC with appropriate
ADME properties. In this study, in an attempt to develop novel NA 150-cavity binders, we
intended to modify the C-5-NH2 group of OC by incorporating pyrazole moiety. A series
of pyrazole-containing C-5-NH2-acyl OC analogues were prepared, and their inhibitory
activities to NA of a clinically isolated A/H3N2 influenza virus strain from Hong Kong
were evaluated.
2. Results and Discussion
2.1. Design of Novel Oseltamivir Analogues
The reported co-crystal structure of OC in complex with NA (PDB: 2HU0) showed
that C-5-NH2 of OC is adjacent to the 150-cavity, which suggests that the C-5-NH2 may
become a modification site towards the discovery of novel NA inhibitors. Based on this
information, attempts on the development of 150-cavity binders as OC inhibitors with
increased activity or overcome drug resistance were carried out. Herein, we designed,
synthesized, and tested a series of C-5-NH2-acyl analogues of OC containing the pyrazole
moiety through an amide bond with different substitutions on pyrazole (Figure 3).
Figure 3. Design of the pyrazole-based C-5-NH2-acyl oseltamivir analogues.
2.2. Chemistry
The pyrazole-based C-5-NH2-acyl OC derivatives were synthesized following the
synthetic routes described in Schemes 1–4.
Scheme 1. Synthetic route of compounds 6a
–
1h
k
. Reagents and conditions: (
a
) for 3a
–
g
:
1a
–
g
, K2CO3,
0
acetone, reflux, overnight, 60–90%; for 3h
–
k
:
–
k
, CuI, trans-N,N -dimethyl-1,2-cyclohexanediamine,
◦
◦
K2CO3, Tol., 110 C, 24 h, 30–55%. (b) NaOH, MeOH/H2O, 50 C; (
c
) oseltamivir phosphate, HATU,
◦
DIPEA, DCM, rt, 60–90%; (d) NaOH, MeOH/H2O, 50 C, 40–70%.

Pharmaceuticals 2021, 14, 371
4 of 20
Scheme 2. Synthetic route of compounds 12a
–
f
.
Reagents and conditions: (
H2O, AcOH, 90 ◦C, 4 h, 70–80%; (
) oseltamivir phosphate, HATU, DIPEA, DCM, rt, 40–80%; (e
a
) Dimethyl ox-
alate, t-BuOK, Tol, rt; (
b
) NH2NH2
d
·
c
) NaOH, MeOH/H2O,
50 ◦C, overnight, 80–90%; (
) NaOH,
◦
MeOH/H2O, 50 C, 50–70%.
Scheme 3. Synthetic route of compounds 16a-c. Reagents and conditions: (a) R3Br, K2CO3, acetone,
◦
reflux, 60–70%; (
b
) NaOH, MeOH/H2O, 50 C, 80–90%; (
c) oseltamivir phosphate, HATU, DIPEA,
◦
DCM, rt, 40–90%; (d) NaOH, MeOH/H2O, 50 C, 40–60%.
As shown in Scheme 1, we first prepared a series of 1H-pyrazole-4-carboxamide
derivatives 6a . Ethyl 1H-pyrazole-4-carboxylate 2 reacted with various aryl bromides
in the presence of K2CO3 [21] to furnish 3a . The arylated derivatives 3h were pre-
pared by the coupling of aryl iodides with compound 2 catalyzed by the CuI-diamine
system with K2CO3 as the base [21]. Hydrolysis of the esters 3a occurred under ba-
sic conditions to afford 1H-pyrazole-4-carboxylic acid derivatives 4a . The condensa-
tion of oseltamivir phosphate with substituted pyrazolecarboxylic acids 4a using the
. Finally, hydrolysis of ethyl ester gave com-
–
k
–
g
–k
–
k
–
k
–
k
HATU/DIPEA methodology provided 5a
–k
pounds 6a–k.
The 1H-pyrazole-5-carboxamide derivatives 12a
in Scheme 2. Treatment of methyl ketones 7a with dimethyl oxalate in the presence of
potassium tert-butoxide afforded intermediates 8a , which were subjected to cyclization
with hydrazine at 50 ◦C in AcOH to give methyl 1H-pyrazole-5-carboxylate derivatives
–f were synthesized as illustrated
–
f
–f
9a
10a
–
–
f
[
f
22], followed by hydrolysis to furnish the 1H-pyrazole-5-carboxylic acid derivatives
. The condensation of 10a with oseltamivir phosphate under the HATU/DIPEA
. After hydrolysis of ethyl ester, compounds 12a were obtained.
–f
condition provided 11a
–f
–f
Alkylation at the pyrazole N-1 position of methyl 3-(4-chlorophenyl)-1H-pyrazole-5-
carboxylate (9b) was accomplished as outlined in Scheme 3. Compound 9b was respectively
alkylated with several alkyl bromides with diverse ring sizes in the presence of K2CO3,
followed by hydrolysis of the esters to afford carboxylic acids 14a
sation of acids 14a with oseltamivir phosphate gave compounds 15a
facile hydrolysis to provide compounds 16a–c.
–
c
. Subsequently, conden-
–
c
–c, which underwent

Pharmaceuticals 2021, 14, 371
5 of 20
Scheme 4. Synthetic route of compounds 24a
–
e
and 26a
HCl, EtOH, reflux, 5 h, 19a
) HATU, DIPEA, DCM, rt, 50–80%; (e
–
d
. Reagents and conditions: (
: 40–80%, 20a
) NaOH, MeOH/H2O,
a
) Dimethyl
oxalate, t-BuOK, Tol, rt; ( ◦
b
) R4NHNH2
·
–
e
–d: 15–30%;
(c
) NaOH, MeOH/H2O, 50 C, 80–90%; (
d
◦
50 C, 40–70%.
1H-pyrazole-3-carboxamide derivatives 24a
–e and 1H-pyrazole-5-carboxamide deriva-
tives 26a were prepared as shown in Scheme 4. Claisen condensation of commercially
–d
available 1-cyclopropylethan-1-one 17 with dimethyl oxalate afforded diketoester 18, which
underwent cyclization with substituted hydrazines to give both the corresponding 1,5-
disubstituted pyrazole-3-carboxylic acid esters 19a
disubstituted pyrazole-5-carboxylic acid esters 20a
purified by column chromatography [23 24]. The esters were hydrolyzed by aqueous
sodium hydroxide solution to afford the corresponding carboxylic acids 21a–e and 22a–d.
–
e, and the isomeric derivatives, 1,3-
–
d. The latter could be isolated and
,
These carboxylic acids reacted with oseltamivir phosphate to furnish compounds 23a
and 25a–d, respectively. After hydrolysis, compounds 24a–e and 26a–d were obtained.
–e
2.3. Neuraminidase Enzyme Inhibitory Assay
A clinically isolated A/H3N2 strain from Hong Kong was cultivated with host cell
cultures followed by lysis. All of the newly synthesized 29 compounds were evaluated for
their inhibitory activity to A/H3N2 NA with OC as the reference drug [17]. The results
were shown in Table 1.

Pharmaceuticals 2021, 14, 371
6 of 20
Table 1. Inhibitory rates of designed compounds against A/H3N2 influenza NA a.
Derivatives
Cpd.
OC
R
10 µM
Cpd.
6f
R
10 µM
\
97.46%
26.79%
6a
32.02%
18.88%
6g
6h
21.72%
38.37%
6b
6c
23.88%
12.26%
6i
6j
52.31%
34.15%
6d
6e
23.37%
42.81%
6k
25.67%
46.7%
12a
12d
12b
12c
50.43%
56.45%
12e
12f
42.02%
40.02%
16a
16b
31.50%
39.81%
16c
48.50%
24a
24b
30.57%
60.91%
24d
24e
42.68%
44.47%
24c
34.75%

Pharmaceuticals 2021, 14, 371
7 of 20
Table 1. Cont.
10 µM
Derivatives
Cpd.
26a
R
Cpd.
26c
R
10 µM
30.41%
31.53%
26b
72.80%
26d
37.17%
A
x−A
A −A
(
bg)−(
0
)
0
bg
a
(%)Inhibitory rate
=
×
100%, Ao is the absorbance of the control (no drug control), Abg is the absorbance of the
A −A
bg
background, Ax is the absorbance of the sample or control compound (OC).
Regarding the first series 6a
compounds displayed mild NA inhibitory activity. Overall, compounds 6a
benzyl or substituted benzyl groups showed less activity than the derivatives 6h
–
k
with a 1H-pyrazole-4-carboxamide moiety, most of the
containing
with
–g
–
k
substituted phenyls. Compound 6i with a 4-methylphenyl group substituted at pyrazole N-
1 position exhibited the highest NA inhibitory activity of 52.31%. Interestingly, compound
6j with a strong electron-donating group (OCH3) and 6k with a weak electron-withdrawing
group (Cl) at the 4-position of the phenyl ring showed inferior inhibitory activities to
6i. These results suggest that both steric and electronic properties need to be further
investigated to clarify the properties for binding to the 150-cavity of NA.
For 1H-pyrazole-5-carboxamide derivatives 12a
ing inhibitory activity at 10 M. The inhibitory activities of the compounds increased with
the electron-withdrawing capability of the halide groups at 4-position of the phenyl ring
12c 12b 12a 12d 12e). Compound 12c with the 4-fluorophenyl group was the most
–f, all the derivatives showed interest-
µ
(
>
>
;
>
active compound in this series, with the percentages of inhibition of 56.45%.
The alkylation of the N-1 position of the pyrazole moiety of compound 12b provided
compounds 16a–c. Results demonstrated that the cyclohexylmethyl group at N-1 of pyza-
role (16c) was preferred. 16c showed similar inhibitory activity to 12b, and the decreased
ring sizes in 16a and 16b caused inferior activities.
1H-pyrazole-3-carboxamide derivatives 24a
carboxamide derivatives 26a displayed diverse NA inhibitory activities from 30% to
72%. When the substituents at para- or meta- positions (e.g., 24c 26c, and 26d), the
inhibitory potencies were similar to the non-substituted benzene derivatives 24a or 26a
Compounds 24b and its isomer 26b with 2-methylphenyl substituted on the pyrazole ring
exhibited the most potent inhibitory activity of 60.91% and 72.80% at 10 M, respectively.
–e and their isomers 1H-pyrazole-5-
–
d
–e,
.
µ
The outstanding inhibitory activity of both compounds suggests that the ortho effect of the
substitutions might be beneficial to the activity.
Briefly, the structure-activity relationship (SAR) discussed above showed that 1H-
pyrazole-5-carboxamide or 1H-pyrazole-3-carboxamide were the optimal linker frag-
ments, and they provided different electronic properties from the substitutions to the
N atom of the pyrazole. Together with our previous study [17], the SAR obtained here will
guide the further design of more potent NA inhibitors.
Overall, most of the pyrazole-based C-5-NH2-acyl analogues of OC inhibited the
enzyme activity of A/H3N2 NA. Compounds 6i
active compounds in each series. Their IC50 values against NA were then determined.
As shown in Table 2, compounds 6i 12c 24b, and 26b exhibited moderate NA inhibitory
activity, among which compound 12c and 26b displayed the best NA inhibitory activity
, 12c, 24b, and 26b represented the most
,
,

Pharmaceuticals 2021, 14, 371
8 of 20
with an IC50 value of 6.98 ± 0.08 µM and 12.27 ± 0.14 µM, respectively. Interestingly, 24b
and 26b against NA at 10 µM exhibited to be more potent than 12c, but compound 12c
inhibited NA with a lower IC50 value, probably because 12c may elicit stronger inhibitory
activity at lower concentrations.
Table 2. Inhibitory activity of 6i, 12c, 24b, and 26b against clinically isolated A/H3N2 NA (µM) a.
OC b
No.
6i
12c
24b
26b
IC50 (µM) a
1.04 ± 0.01
27.56 ± 0.55
6.98 ± 0.08
26.99 ± 0.31
12.27 ± 0.14
a
IC50 is the compound concentration producing 50% inhibition of NA, the values are the mean of three indepen-
dent experiments. IC50 unit of OC is nM.
b
2.4. In Silico ADME Prediction
To further explore the druggability of these OC derivatives, the ADME properties
were predicted by Qikprop (Maestro 10.2) [17]. The ADME descriptors of compounds
6i, 12c, 24b, and 26b were calculated, including octanol-water partitioning coefficient
(QPlogPo/w), aqueous solubility (QPlogS), binding to human serum albumin (QPlogKhsa),
brain/blood partition coefficient (QPlogBB), number of likely metabolic reactions (#metab),
central nervous system activity (CNS), predicted IC50 value for the blockage of HERG K+
channels (QPloghERG), apparent Caco-2 cell permeability (QPPCaco), apparent MDCK
cell permeability (QPPMDCK), and human oral absorption.
As shown in Table 3, the four C5-NH-acyl derivatives were predicted to be signifi-
cantly more lipophilic with lower aqueous solubility in comparison with OC. Moreover,
the percentages of human oral absorption of the new derivatives were 60~70%, higher than
32.7% for OC. Meanwhile, QPPCaco and QPPMDCK of the derivatives were predicted to
be 3- or 7-fold higher than that of OC. These data suggest that our derivatives could be well
absorbed by the intestine and thereof could be orally administrated directly instead of con-
verting to the ester prodrug as OC. Other parameters such as QPlogBB, CNS, QPloghERG,
and #metab remained within the standard ranges which suggests these derivatives should
be metabolically stable and nontoxic to both the nervous system and heart.
Table 3. Comparison of computational pharmacokinetic parameters (ADME) of OC derivatives.
Principal
Descriptors
Standard
Range *
OC
6i
12c
24b
26b
QPlogPo/w
QPlogS
QPlogKhsa
QPlogBB
#metab
−1.654
−1.164
−0.873
−1.05
3
3.361
−5.741
−0.043
−2.186
3
3.337
−5.931
−0.036
−2.186
2
4.106
−6.323
0.277
−2.086
3
4.380
−6.648
0.291
−1.714
3
−2.0–6.5
−6.5–0.5
−1.5–1.5
−3.0–1.2
1–8
−
2 (inactivity),
CNS
−2
−1.322
7.34
−2
−3.348
20.787
−2
−2.975
17.378
−2
−3.006
22.727
−2
−3.085
54.32
+2 (activity)
<−5
QPloghERG
QPPCaco
<25 poor,
>500 great
<25 poor,
>500 great
QPPMDCK
6.146
32.756
Middle
17.413
70.213
Middle
19.348
68.675
Middle
20.759
62.305
Middle
42.816
70.687
Middle
(%) Human Oral
Absorption
Human Oral
Absorption
<25% is poor,
>80% is high
* Statistics of 95% of known drugs according to Qikprop (Maestro 10.2).
2.5. Molecular Docking Studies
As described previously, molecular docking studies were performed to reveal the
potential interactions between NA and the newly designed OC derivatives. All the final
compounds were docked with the crystal structure of NA (PDB: 2HU0) using Glide of

Pharmaceuticals 2021, 14, 371
9 of 20
Maestro 10.2 [17]. Results showed that most of the derivatives fitted into the NA active site
with their OC motif and occupied the 150-cavity with the substituted pyrazole moieties as
designed (Figure S1 and Figure 4A).
Figure 4. (A) Binding positions of compounds 12c (green) and 26b (cyan) to NA (PDB: 2HU0); Detailed interactions of 12c
(B) and 26b (C) with NA: Hydrogen/ionic bonding interactions (green) and hydrophobic interactions (magenta).
Details of the interactions between compound 12c and NA are shown in Figure 4B
and the key H-bonds were summarized in Table 4. The OC part of 12c formed hydrogen
bonding and hydrophobic interactions with the NA active site, including hydrogen bonds
between the carboxylic acid group and residues Asn294 and Ser246, and hydrophobic in-
teractions between the pentan-3-yloxy group and residues Arg152 and Trp178. In addition,
the 5-(3-(4-fluorophenyl)-1H-pyrazole-5-carboxamide side chain formed hydrogen bonds
with residues Arg371 and Arg430, and hydrophobic interaction with Pro431.
Table 4. Hydrogen bonding interactions between NA (PDB: 2HU0) and compounds 12c and 26b.
H-Bonding between NA and 12c
H-Bonding between NA and 26b
Hydrogen Bonds
Hydrogen Bonds
Distance (Å)
Distance (Å)
SER246: HG/12c: O9
ASN294: HD21/12c: O8
ARG371: HH11/12c: O23
ARG371: HH21/12c: O23
ARG371: HH21/12c: N27
ARG430: HH22/12c: F34
2.10
3.15
2.24
1.95
2.67
2.93
ARG156: HH11/26b: O19
ARG156: HH21/26b: O19
ARG292: HH21/26b: O9
ARG371: HH11/26b: O9
ARG371: HH21/26b: O9
26b: H59/ASP151: OD1
1.82
2.67
1.92
1.57
3.01
2.71
26b interacts with some different residues in the NA active site when compared with
compound 12c (Figure 4C, Table 4). The carboxylic group formed ionic bonding interactions
with residue Arg292 and Arg371, and hydrogen bonding with Tyr406. Additionally, the C-4
amide group contacted Arg156 via two hydrogen bonds. These changes might enhance the
stability of the 12c-NA complex. Finally, hydrophobic interactions were formed between
the phenyl ring and residues Pro431 and Arg430. Overall, the additional interactions of
OC derivatives with NA may improve the binding affinity.
3. Materials and Methods
3.1. Biology
NA Enzyme Inhibitory Assay
The NA inhibitory assay was performed using the commercially available NA-Fluor
™
Influenza Neuraminidase Assay Kit as described previously [17].
3.2. Chemistry
3.2.1. General Methods
Starting materials and reagents, unless otherwise stated, were commercial grade and
used without further purification. Thin-layer chromatography (TLC) on glass sheets (Silica

Pharmaceuticals 2021, 14, 371
10 of 20
gel F254) was visualized under UV light and used to monitor all the reactions. Column
chromatography was carried out using silica gel (200–300 mesh). Compound structures
1
were confirmed by H NMR (400 MHz) and 13C NMR (100 MHz) spectra which were
acquired using a BRUKER AVANCE-III spectrometer. NMR chemical shifts were given in
δ
(ppm) and coupling constants (J) in Hz. The solvents for NMR were DMSO-d6 (δ 2.50
for 1H) or chloroform ( 7.26 for 1H). High-resolution MS spectra were measured using a
δ
Micromass® Q-ToF2 mass spectrometer.
3.2.2. General Procedures for the Synthesis of Compounds 3a–k
For compounds 3a–g: A mixture of ethyl-4-pyrazole carboxylate (280 mg, 2 mmol),
K2CO3 (1.38 g, 10 mmol), and a bromomethyl arene (2.4 mmol) in acetone (10 mL) was
heated to reflux for 12 h. After cooling to room temperature, K2CO3 was filtered off and
the filtrate was concentrated, purified by column chromatography, eluting with a gradient
of hexane/ethyl acetate (15:1 to 6:1) to provide compounds 3a–g.
For compounds 3h–3k: To a Schlenk tube were added ethyl-4-pyrazole carboxylate
(280 mg, 2 mmol), an aryl iodide (2.2 mmol), CuI (76 mg, 0.4 mmol), K2CO3 (580 mg,
4.2 mmol) and trans-N,N0-dimethyl-1,2-cyclohexanediamine (113 mg, 0.8 mmol) and
toluene (5 mL). The reaction tube was charged with N2 and immersed in a preheated
◦
oil bath for stirring at 110 C for 24 h. The reaction mixture was concentrated, and the
resulting residue was purified by column chromatography, eluting with a gradient of
hexane/ethyl acetate (20:1 to 10:1) to provide the desired products.
3.2.3. General Procedure for the Synthesis of Compounds 4a–k
Each ethyl ester of 3a–k was dissolved in MeOH (10 mL), 1 M NaOH aqueous solution
(2 mL) was added. The mixture was stirred at 50 ◦C overnight. The solvent was removed
under reduced pressure and the residue was diluted with 10 mL water and washed with
DCM. The aqueous phase was acidified by 1 M HCl to precipitate the product which was
collected by filtration and dried in vacuo. The products were used directly for the next step
of the reaction.
3.2.4. General Procedure for the Synthesis of Compounds 5a–k
A mixture of oseltamivir phosphate (82 mg, 0.2 mmol), each pyrazole-4-carboxylic acid
derivative of 4a–k (0.24 mmol), HATU (115 mg, 0.3 mmol) and DIPEA (83 µL, 0.5 mmol) in
DCM (10 mL) was stirred at room temperature overnight. The reaction was quenched by
saturated NaHCO3 aqueous solution and the resulting mixture was extracted with DCM
(20 mL
×
3). The combined organic phases were washed with brine, dried over Na2SO4,
concentrated, and purified by silica gel column chromatography, eluting with a gradient of
DCM/MeOH (50:1 to 30:1) to afford the titled compound.
3.2.5. General Procedure for the Synthesis of Compounds 6a–k
Each compound of 5a
aqueous solution (2 mL) was added. The mixture was heated to 50 C overnight. The
solvent was removed under reduced pressure and the residue was diluted with 10 mL
–
k
(0.1 mmol) was dissolved in MeOH (10 mL), 1 M NaOH
◦
water and washed with DCM (10 mL
precipitate the product which was collected by filtration and dried in vacuo.
×
2). The aqueous phase was acidified by 1 M HCl to
3.2.6. (3R,4R,5S)-4-acetamido-5-(1-benzyl-1H-pyrazole-4-carboxamido)-3-(pentan-3-
yloxy) cyclohex-1-ene-1-carboxylic acid (6a)
◦
Yield 59%; white solid, mp 273–275 C. 1H NMR (400 MHz, DMSO-d6)
δ 12.60 (s, 1H),
8.18 (s, 1H), 7.97–7.80 (m, 2H), 7.79 (s, 1H), 7.42–7.28 (m, 3H), 7.25 (d, J = 6.7 Hz, 2H), 6.63
(s, 1H), 5.35 (s, 2H), 4.08 (ddd, J = 19.4, 17.3, 9.0 Hz, 2H), 3.83 (dd, J = 19.9, 8.9 Hz, 1H),
3.45–3.30 (m, 1H), 2.55 (d, J = 5.1 Hz, 1H), 2.38–2.24 (m, 1H), 1.68 (s, 3H), 1.53–1.30 (m, 4H),
0.84 (t, J = 7.3 Hz, 3H), 0.77 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ
170.3,
167.9, 162.0, 139.2, 138.3, 137.4, 131.8, 129.8, 129.0, 128.3, 128.2, 119.1, 81.6, 75.5, 55.5, 54.6,

Pharmaceuticals 2021, 14, 371
11 of 20
48.3, 30.8, 26.3, 25.8, 23.2, 9.8, 9.5. HRMS (ESI): calcd for C25H31N4O5, [M-H]− 467.2300,
found 467.2309.
3.2.7. (3R,4R,5S)-4-acetamido-5-(1-(2-chlorobenzyl)-1H-pyrazole-4-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (6b)
Yield 52%; white solid, mp 265–267 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.16 (s, 1H),
7.94–7.83 (m, 2H), 7.82 (s, 1H), 7.50 (dd, J = 7.6, 1.4 Hz, 1H), 7.42–7.31 (m, 2H), 7.12 (dd,
J = 7.3, 1.8 Hz, 1H), 6.63 (s, 1H), 5.46 (s, 2H), 4.14 (d, J = 8.1 Hz, 1H), 4.11–4.02 (m, 1H), 3.84
(dd, J = 20.0, 9.0 Hz, 1H), 3.45–3.30 (m, 1H), 2.57–2.51 (m, 1H), 2.32 (dd, J = 17.5, 10.4 Hz,
1H), 1.68 (s, 3H), 1.52–1.30 (m, 4H), 0.84 (t, J = 7.3 Hz, 3H), 0.77 (t, J = 7.3 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 170.3, 167.7, 161.9, 139.4, 138.2, 134.6, 132.9, 132.2, 130.8, 130.4, 130.0,
129.8, 128.0, 119.1, 81.6, 75.6, 54.6, 53.2, 48.4, 30.8, 26.3, 25.8, 23.2, 9.8, 9.5. HRMS (ESI): calcd
for C25H30ClN4O5, [M-H]− 501.1910, found 501.1912.
3.2.8. (3R,4R,5S)-4-acetamido-5-(1-(3-chlorobenzyl)-1H-pyrazole-4-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (6c)
◦
Yield 63%; white solid, mp 245–247 C. 1H NMR (400 MHz, DMSO-d6)
δ 12.62 (s, 1H),
8.21 (s, 1H), 7.84 (dd, J = 12.9, 9.2 Hz, 2H), 7.80 (s, 1H), 7.44–7.34 (m, 2H), 7.29 (s, 1H),
7.25–7.17 (m, 1H), 6.63 (s, 1H), 5.38 (s, 2H), 4.17–4.01 (m, 2H), 3.83 (dd, J = 19.9, 9.1 Hz, 1H),
3.45–3.30 (m, 1H), 2.55–2.51 (m, 1H), 2.32 (dd, J = 17.6, 10.4 Hz, 1H), 1.67 (s, 3H), 1.53–1.34
(m, 4H), 0.84 (t, J = 7.3 Hz, 3H), 0.76 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ
170.2, 167.7, 161.9, 139.9, 139.4, 138.2, 133.6, 132.0, 131.0, 129.8, 128.2, 128.0, 126.0, 119.3, 81.6,
75.6, 54.7, 54.6, 48.4, 30.8, 26.3, 25.8, 23.2, 9.8, 9.5. HRMS (ESI): calcd for C25H30ClN4O5,
[M-H]− 501.1910, found 501.1912.
3.2.9. (3R,4R,5S)-4-acetamido-5-(1-(3-nitrobenzyl)-1H-pyrazole-4-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (6d)
Yield 59%; white solid, mp 236–238 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.28 (s, 1H),
8.22–8.15 (m, 1H), 8.12 (s, 1H), 7.94–7.79 (m, 3H), 7.71 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 7.8 Hz,
1H), 6.63 (s, 1H), 5.54 (s, 2H), 4.17–4.01 (m, 2H), 3.84 (dd, J = 19.9, 9.0 Hz, 1H), 3.45–3.30
(m, 1H), 2.60–2.51 (m, 1H), 2.32 (dd, J = 17.5, 10.4 Hz, 1H), 1.68 (s, 3H), 1.51–1.31 (m, 4H),
0.84 (t, J = 7.3 Hz, 3H), 0.76 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.3,
167.8, 161.9, 148.3, 139.7, 139.6, 138.1, 134.9, 132.3, 130.7, 129.9, 123.3, 122.8, 119.4, 81.6, 75.6,
54.6, 54.4, 48.4, 30.8, 26.3, 25.7, 23.1, 9.8, 9.5. HRMS (ESI): calcd for C25H30N5O7, [M-H]−
512.2151, found 512.2156.
3.2.10. (3R,4R,5S)-4-acetamido-5-(1-(3-methoxybenzyl)-1H-pyrazole-4-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (6e)
Yield 62%; white solid, mp 259–261 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.17 (s, 1H),
7.88 (d, J = 9.1 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.79 (s, 1H), 7.27 (t, J = 7.8 Hz, 1H), 6.88 (dd,
J = 8.1, 2.1 Hz, 1H), 6.84–6.74 (m, 2H), 6.63 (s, 1H), 5.32 (s, 2H), 4.18–4.00 (m, 2H), 3.83 (dd,
J = 20.0, 9.0 Hz, 1H), 3.73 (s, 3H), 3.45–3.30 (m, 1H), 2.59–2.50 (m, 1H), 2.37–2.27 (m, 1H),
1.67 (s, 3H), 1.51–1.28 (m, 4H), 0.84 (t, J = 7.3 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 170.2, 167.7, 162.0, 159.8, 139.2, 138.9, 138.3, 131.8, 130.2, 129.8, 120.3,
119.1, 114.0, 113.6, 81.6, 75.6, 55.5, 55.4, 54.6, 48.3, 30.8, 26.3, 25.7, 23.1, 9.8, 9.5. HRMS (ESI):
calcd for C26H33N4O6, [M-H]− 497.2406, found 497.2410.
3.2.11. (3R,4R,5S)-4-acetamido-5-(1-(4-methoxybenzyl)-1H-pyrazole-4-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (6f)
Yield 45%; white solid, mp 269–271 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.11 (s, 1H),
7.87 (d, J = 9.1 Hz, 1H), 7.79 (d, J = 8.9 Hz, 1H), 7.76 (s, 1H), 7.24 (d, J = 8.5 Hz, 2H), 6.92
(d, J = 8.5 Hz, 2H), 6.63 (s, 1H), 5.26 (s, 2H), 4.14 (d, J = 8.0 Hz, 1H), 4.10–4.00 (m, 1H),
3.83 (dd, J = 19.9, 9.0 Hz, 1H), 3.74 (s, 3H), 3.45–3.30 (m, 1H), 2.63–2.45 (m, 1H), 2.34–2.20
(m, 1H), 1.68 (s, 3H), 1.53–1.30 (m, 4H), 0.84 (t, J = 7.3 Hz, 3H), 0.77 (t, J = 7.3 Hz, 3H). 13
C

Pharmaceuticals 2021, 14, 371
12 of 20
NMR (100 MHz, DMSO-d6)
δ
170.3, 167.7, 162.0, 159.4, 139.0, 138.2, 131.4, 129.9, 129.8, 129.2,
119.0, 114.4, 81.6, 75.6, 55.6, 55.0, 54.6, 48.3, 30.8, 26.3, 25.7, 23.2, 9.8, 9.5. HRMS (ESI): calcd
for C26H33N4O6, [M-H]− 497.2408, found 497.2410.
3.2.12. (3R,4R,5S)-4-acetamido-3-(pentan-3-yloxy)-5-(1-(4-(trifluoromethyl)
benzyl)-1H-pyrazole-4-carboxamido) cyclohex-1-ene-1-carboxylic acid (6g)
Yield 51%; white solid, mp 259–261 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.24 (s, 1H),
7.86 (dd, J = 15.2, 10.8 Hz, 3H), 7.73 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 6.64 (s, 1H),
5.48 (s, 2H), 4.22–4.01 (m, 2H), 3.84 (dd, J = 19.8, 9.1 Hz, 1H), 3.40–3.30 (m, 1H), 2.60–2.51
(m, 1H), 2.32 (dd, J = 17.5, 10.4 Hz, 1H), 1.68 (s, 3H), 1.53–1.31 (m, 4H), 0.84 (t, J = 7.3 Hz,
3H), 0.77 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.3, 167.7, 161.9, 142.2, 139.5,
138.3, 132.3, 129.7, 128.85 (d, J = 31.7 Hz), 128.83, 126.0 (d, J = 3.2 Hz), 125.9 (d, J = 3.2 Hz),
124.6 (q, J = 260.3 Hz), 119.3, 81.6, 75.6, 54.8, 54.56, 48.4, 30.8, 26.3, 25.7, 23.2, 9.8, 9.5. HRMS
(ESI): calcd for C26H30F3N4O5, [M-H]− 535.2174, found 535.2178.
3.2.13. (3R,4R,5S)-4-acetamido-3-(pentan-3-yloxy)-5-(1-(o-tolyl)-1H-pyrazole-4-
carboxamido) cyclohex-1-ene-1-carboxylic acid (6h)
Yield 61%; white solid, mp 253–255 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.34 (s, 1H),
8.02 (s, 1H), 7.93 (dd, J = 9.0, 4.2 Hz, 2H), 7.47–7.32 (m, 4H), 6.65 (s, 1H), 4.22–4.05 (m, 2H),
3.87 (dd, J = 20.0, 9.0 Hz, 1H), 3.45–3.30 (s, 1H), 2.58 (dd, J = 17.6, 5.0 Hz, 1H), 2.34 (dd,
J = 17.4, 10.4 Hz, 1H), 2.21 (s, 3H), 1.71 (s, 3H), 1.52–1.34 (m, 4H), 0.85 (t, J = 7.3 Hz, 3H),
0.78 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.4, 167.7, 161.9, 140.0, 139.6,
138.3, 133.2, 132.8, 131.8, 129.8, 129.2, 127.3, 126.2, 119.8, 81.6, 75.6, 54.7, 48.5, 30.8, 26.3, 25.8,
23.2, 18.2, 9.9, 9.5. HRMS (ESI): calcd for C25H31N4O5, [M-H]− 467.2300, found 467.2304.
3.2.14. (3R,4R,5S)-4-acetamido-3-(pentan-3-yloxy)-5-(1-(p-tolyl)-1H-pyrazole-4-
carboxamido) cyclohex-1-ene-1-carboxylic acid (6i)
Yield 45%; white solid, mp 282–284 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.79 (s, 1H),
8.03 (s, 1H), 7.98 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 9.1 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H),
7.33 (d, J = 8.1 Hz, 2H), 6.66 (s, 1H), 4.25–4.07 (m, 2H), 3.87 (dd, J = 19.8, 9.1 Hz, 1H),
3.45–3.30 (m, 1H), 2.58 (dd, J = 17.5, 4.8 Hz, 1H), 2.45–2.30 (m, 1H), 2.35 (s, 3H), 1.70 (s, 3H),
1.53–1.33 (m, 4H), 0.85 (t, J = 7.3 Hz, 3H), 0.78 (t, J = 7.3 Hz, 3H), 0.67–0.66 (m, 1H). 13
C
NMR (100 MHz, DMSO-d6) δ 170.3, 167.7, 161.7, 140.4, 138.3, 137.4, 136.9, 130.5, 129.8, 129.0,
120.8, 119.1, 81.6, 75.6, 54.7, 48.4, 30.8, 26.3, 25.8, 23.2, 20.9, 9.9, 9.5. HRMS (ESI): calcd for
C25H31N4O5, [M-H]− 467.2300, found 467.2306.
3.2.15. (3R,4R,5S)-4-acetamido-5-(1-(4-methoxyphenyl)-1H-pyrazole-4-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (6j)
Yield 47%; white solid, mp 277–279 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.72 (s, 1H),
8.01 (s, 1H), 7.94 (dd, J = 14.9, 9.2 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.7 Hz, 2H),
6.65 (s, 1H), 4.20–4.08 (m, 2H), 3.93–3.85 (m, 1H), 3.80 (s, 3H), 3.45–3.30 (m, 1H), 2.57 (dd,
J = 17.4, 4.3 Hz, 1H), 2.41–2.30 (m, 1H), 1.70 (s, 3H), 1.51–1.33 (m, 4H), 0.85 (t, J = 7.2 Hz,
3H), 0.78 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.3, 167.8, 161.8, 158.6,
140.2, 138.1, 133.2, 130.0, 128.9, 120.8, 120.6, 115.2, 81.6, 75.6, 55.9, 54.7, 48.4, 30.9, 26.3, 25.8,
23.2, 9.9, 9.5. HRMS (ESI): calcd for C25H31N4O6, [M-H]− 483.2249, found 483.2252.
3.2.16. (3R,4R,5S)-4-acetamido-5-(1-(4-chlorophenyl)-1H-pyrazole-4-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (6k)
Yield 55%; white solid, mp 295–297 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 8.87 (s, 1H),
8.08 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 9.3 Hz, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.60
(d, J = 8.6 Hz, 2H), 6.65 (s, 1H), 4.22–4.07 (m, 2H), 3.87 (dd, J = 19.7, 9.3 Hz, 1H), 3.45–3.30
(m, 1H), 2.57 (dd, J = 17.5, 4.7 Hz, 1H), 2.41–2.30 (m, 1H), 1.70 (s, 3H), 1.52–1.33 (m, 4H),
0.85 (t, J = 7.2 Hz, 3H), 0.77 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ
170.3,
167.7, 161.5, 140.8, 138.4, 138.3, 131.6, 130.1, 129.8, 129.5, 121.3, 120.9, 81.6, 75.6, 54.7, 48.5,

Pharmaceuticals 2021, 14, 371
13 of 20
30.8, 26.3, 25.7, 23.2, 9.9, 9.5. HRMS (ESI): calcd for C24H28ClN4O5, [M-H]− 487.1754,
found 487.1759.
3.2.17. General Procedure for the Synthesis of Compounds 9a–f
To a stirred solution of dimethyl oxalate (1.30 g, 11 mmol) and an aryl methyl ketone
(10 mmol) in toluene (60 mL) was added a solution of potassium tert-butoxide (1.35 g,
12 mmol) in THF (60 mL). The resulting solution was stirred at room temperature overnight.
The reaction was quenched with 1 N HCl solution and extracted with ethyl acetate. The
combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced
pressure to provide the crude products 8a–f.
The crude product was dissolved in AcOH (10 mL) and hydrazine hydrate (534 µL,
10 mmol) was added. The solution was heated to 90 ◦C for 4 h. The solvent was removed
under reduced pressure and the residue was dispersed in EtOAc. The precipitate was
collected via filtration and washed by a small amount of EtOAc to afford compounds 9a–f
without further purification.
3.2.18. General Procedure for the Synthesis of Compounds 10a–f
The preparation of 10a–f was performed similarly as described for compounds 4a–k.
3.2.19. General Procedure for the Synthesis of Compounds 11a–f
The condensation of 10a–f with oseltamivir phosphate to afford 11a–f was performed
in a similar manner as described for compounds 5a–k.
3.2.20. General Procedure for the Synthesis of Compounds 12a–f
Each methyl ester of 11a
solution (2 mL) was added. The mixture was stirred at 50 C overnight. The solvent
was removed under reduced pressure and the residue was diluted with 10 mL water and
–
f
was dissolved in MeOH (10◦mL), 1 M NaOH aqueous
washed with DCM (10 mL
the product which was collected by filtration and dried in vacuo.
×
2). The aqueous phase was acidified by 1 M HCl to precipitate
3.2.21. (3R,4R,5S)-4-acetamido-3-(pentan-3-yloxy)-5-(3-phenyl-1H-pyrazole-5-
carboxamido) cyclohex-1-ene-1-carboxylic acid (12a)
◦
Yield 69%; white solid, mp 234–236 C. 1H NMR (400 MHz, DMSO-d6)
δ 8.00 (d, J = 8.6 Hz,
2H), 7.80 (d, J = 7.5 Hz, 2H), 7.46 (t, J = 7.4 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 7.08 (s, 1H), 6.64
(s, 1H), 4.18–4.08 (m, 2H), 4.00–3.85 (m, 1H), 3.46–3.30 (m, 1H), 2.67 (d, J = 15.7 Hz, 1H),
2.35 (dd, J = 16.9, 8.8 Hz, 1H), 1.75 (s, 3H), 1.54–1.34 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H), 0.80
(t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.4, 168.3, 161.3, 147.4, 145.4, 136.6,
130.7, 129.4, 128.7, 125.7, 102.8, 81.6, 75.3, 53.5, 48.4, 30.7, 26.3, 25.8, 23.2, 9.8, 9.6. HRMS
(ESI): calcd for C24H29N4O5, [M-H]− 453.2143, found 453.2144.
3.2.22. (3R,4R,5S)-4-acetamido-5-(3-(4-chlorophenyl)-1H-pyrazole-5-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (12b)
◦
Yield 62%; white solid, mp 266–268 C. 1H NMR (400 MHz, DMSO-d6)
δ 13.74 (s, 1H),
8.05 (s, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.12
(s, 1H), 6.67 (s, 1H), 4.25–4.06 (m, 2H), 3.94 (dd, J = 17.6, 8.7 Hz, 1H), 3.45–3.40 (m, 1H),
2.65 (d, J = 14.0 Hz, 1H), 2.38 (dd, J = 16.9, 9.2 Hz, 1H), 1.75 (s, 3H), 1.56–1.31 (m, 4H), 0.86
(t, J = 7.2 Hz, 3H), 0.79 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.4, 167.9,
160.8, 137.4, 133.2, 130.0, 129.5, 127.4, 103.1, 81.6, 75.2, 53.6, 48.4, 30.5, 26.3, 25.8, 23.1, 9.8,
9.6, 9.3. HRMS (ESI): calcd for C24H28ClN4O5, [M-H]− 487.1754, found 487.1761.
3.2.23. (3R,4R,5S)-4-acetamido-5-(3-(4-fluorophenyl)-1H-pyrazole-5-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (12c)
◦
Yield 55%; white solid, mp 259–261 C. 1H NMR (400 MHz, DMSO-d6)
δ 13.46 (s, 1H),
8.02 (s, 1H), 7.96 (d, J = 8.8 Hz, 1H), 7.83 (dd, J = 8.3, 5.5 Hz, 2H), 7.30 (t, J = 8.8 Hz, 2H),

Pharmaceuticals 2021, 14, 371
14 of 20
7.07 (s, 1H), 6.68 (s, 1H), 4.22–4.07 (m, 2H), 3.95 (dd, J = 18.2, 8.8 Hz, 1H), 3.45–3.40 (m, 1H),
2.65 (dd, J = 17.3, 4.1 Hz, 1H), 2.38 (dd, J = 17.5, 9.3 Hz, 1H), 1.75 (s, 3H), 1.54–1.34 (m, 4H),
0.86 (t, J = 7.3 Hz, 3H), 0.79 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.4,
167.8, 162.4 (d, J = 243.9 Hz), 160.9, 145.3, 137.6, 129.8, 127.8, 127.7 (d, J = 8.1 Hz), 116.4
(d, J = 21.6 Hz), 102.8, 81.6, 75.2, 53.5, 48.3, 30.4, 26.3, 25.8, 23.1, 9.8, 9.6. HRMS (ESI): calcd
for C24H28FN4O5, [M-H]− 471.2049, found 471.2051.
3.2.24. (3R,4R,5S)-4-acetamido-3-(pentan-3-yloxy)-5-(3-(4-(trifluoromethyl)
phenyl)-1H-pyrazole-5-carboxamido) cyclohex-1-ene-1-carboxylic acid (12d)
◦
Yield 50%; white solid, mp 270–272 C. 1H NMR (400 MHz, DMSO-d6)
δ 13.86 (s, 1H),
8.13 (s, 1H), 8.02 (s, 1H), 8.00 (s, 1H), 7.96 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.24
(s, 1H), 6.68 (s, 1H), 4.21–4.09 (m, 2H), 3.95 (dd, J = 18.0, 8.9 Hz, 1H), 3.45–3.30 (m, 1H), 2.65
(dd, J = 17.2, 3.6 Hz, 1H), 2.40 (dd, J = 17.3, 9.4 Hz, 1H), 1.74 (s, 3H), 1.52–1.35 (m, 4H), 0.85
(t, J = 7.3 Hz, 3H), 0.79 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.4, 167.8,
160.7, 137.7, 129.8, 128.71, 128.68 (q, J = 31.7 Hz), 126.4 (q, J = 3.6 Hz), 126.2, 126.0, 123.3,
120.6 (q, J = 270.2 Hz), 103.8, 81.6, 75.3, 53.7, 48.4, 30.4, 26.3, 25.8, 23.1, 9.8, 9.5. HRMS (ESI):
calcd for C25H28F3N4O5, [M-H]− 521.2017, found 521.2023.
3.2.25. (3R,4R,5S)-4-acetamido-5-(3-(4-methoxyphenyl)-1H-pyrazole-5-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (12e)
◦
Yield 59%; white solid, mp 273–275 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.96 (d, J = 8.6 Hz,
2H), 7.72 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 8.7 Hz, 2H), 6.97 (s, 1H), 6.68 (s, 1H), 4.10 (dd,
J = 17.4, 11.7 Hz, 2H), 3.96 (dd, J = 18.2, 8.7 Hz, 1H), 3.79 (s, 3H), 3.45–3.42 (m, 1H), 2.66
(dd, J = 17.5, 4.4 Hz, 1H), 2.36 (dd, J = 17.5, 9.1 Hz, 1H), 1.76 (s, 3H), 1.54–1.35 (m, 4H), 0.86
(t, J = 7.3 Hz, 3H), 0.80 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.4, 167.8,
161.4, 159.8, 145.4, 137.5, 129.9, 127.1, 122.8, 114.8, 102.0, 81.6, 75.2, 55.7, 53.4, 48.3, 30.5, 26.3,
25.8, 23.1, 9.8, 9.6. HRMS (ESI): calcd for C25H31N4O6, [M-H]− 483.2249, found 483.2253.
3.2.26. (3R,4R,5S)-4-acetamido-3-(pentan-3-yloxy)-5-(3-(thiophen-2-yl)-1H-pyrazole-5-
carboxamido) cyclohex-1-ene-1-carboxylic acid (12f)
◦
Yield 65%; white solid, mp 225–227 C. 1H NMR (400 MHz, DMSO-d6)
δ 13.64 (s, 1H),
8.24 (s, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.54 (s, 1H), 7.41 (s, 1H), 7.13 (s, 1H), 6.94 (s, 1H), 6.65
(s, 1H), 4.19–4.05 (m, 2H), 3.95–3.90 (m, 1H), 3.45–3.30 (m, 1H), 2.60 (d, J = 14.5 Hz, 1H),
2.46–2.33 (m, 1H), 1.73 (s, 3H), 1.56–1.35 (m, 4H), 0.86 (t, J = 7.3 Hz, 3H), 0.79 (t, J = 7.2 Hz,
3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.3, 167.9, 160.0, 137.4, 130.1, 128.4, 126.2, 124.6,
102.6, 81.6, 75.3, 53.9, 48.4, 30.5, 26.3, 25.8, 23.6, 9.9, 9.6. HRMS (ESI): calcd for C22H27N4O5S,
[M-H]− 459.1708, found 459.1712.
3.2.27. General Procedure for the Synthesis of Compounds 13a–c
A mixture of methyl 3-(4-chlorophenyl)-1H-pyrazole-5-carboxylate (9b, 237 mg, 1 mmol),
K2CO3 (276 g, 2 mmol), and alkyl bromide (3 mmol) in acetone (10 mL) was heated to
reflux for 12 h. After cooling to room temperature, K2CO3 was filtered off and the filtrate
was concentrated and purified by column chromatography, eluting with hexane/ethyl
acetate (6:1) to provide the titled compound.
3.2.28. General Procedure for the Synthesis of Compounds 14a–c
The hydrolysis of 13a
pounds 4a–k.
–c was performed in a similar manner as described for com-
3.2.29. General Procedure for the Synthesis of Compounds 15a–c
The condensation of 13a with oseltamivir phosphate was performed in a similar
manner as described for compounds 5a–k.
–
c

Pharmaceuticals 2021, 14, 371
15 of 20
3.2.30. General Procedure for the Synthesis of Compounds 16a–c
Each ester of 15a–c was dissolved in MeOH (10 mL), 1 M NaOH aqueous solution
(2 mL) was added. The mixture was stirred at 50 ◦C overnight. The solvent was removed
under reduced pressure and the residue was diluted with 10 mL water and washed with
DCM (10 mL
×
2). The aqueous phase was acidified by 1 M HCl to precipitate the product
which was collected by filtration and dried in vacuo.
3.2.31. (3R,4R,5S)-4-acetamido-5-(3-(4-chlorophenyl)-1-(cyclopropylmethyl)-1H-pyrazole-
5-carboxamido)-3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (16a)
◦
Yield 65%; white solid, mp 269–271 C. 1H NMR (400 MHz, DMSO-d6)
δ 8.42 (d, J = 8.9 Hz,
1H), 8.02 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 7.14 (s, 1H),
6.56 (s, 1H), 4.37 (ddd, J = 45.8, 13.6, 7.2 Hz, 2H), 4.17–4.08 (m, 2H), 3.90–3.80 (m, 1H),
3.45–3.32 (m, 1H), 2.59 (d, J = 13.2 Hz, 1H), 2.44–2.33 (m, 1H), 1.73 (s, 3H), 1.54–1.36 (m, 4H),
1.34–1.28 (m, 1H), 0.85 (t, J = 7.3 Hz, 3H), 0.78 (t, J = 7.2 Hz, 3H), 0.46 (d, J = 7.6 Hz, 2H),
0.33–0.40 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
δ 170.3, 169.3, 159.5, 147.7, 137.4, 135.6,
132.7, 132.0, 129.4, 127.1, 104.9, 81.5, 75.8, 55.2, 54.7, 48.8, 31.1, 26.4, 25.7, 23.2, 12.5, 9.9, 9.5,
3.9, 3.8. HRMS (ESI): calcd for C28H34ClN4O5, [M-H]− 541.2223, found 541.2229.
3.2.32. (3R,4R,5S)-4-acetamido-5-(3-(4-chlorophenyl)-1-(cyclobutylmethyl)-1H-pyrazole-
5-carboxamido)-3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (16b)
◦
Yield 51%; white solid, mp 265–267 C. 1H NMR (400 MHz, DMSO-d6)
δ 8.39 (d, J = 8.8 Hz,
1H), 8.02 (d, J = 8.9 Hz, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.12 (s, 1H), 6.63
(s, 1H), 4.54 (ddd, J = 40.1, 13.1, 7.5 Hz, 2H), 4.20–4.10 (s, 2H), 3.88 (dd, J = 19.3, 9.3 Hz, 1H),
3.45–3.30 (m, 1H), 2.82–2.71 (m, 1H), 2.56 (d, J = 13.6 Hz, 1H), 2.46–2.34 (m, 1H), 1.96–1.85
(m, 2H), 1.85–1.65 (m, 7H), 1.54–1.33 (m, 4H), 0.85 (t, J = 7.0 Hz, 3H), 0.78 (t, J = 7.0 Hz,
3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.2, 168.2, 159.5, 147.6, 137.7, 137.5, 132.7, 132.0,
129.4, 127.0, 104.8, 81.6, 75.6, 55.3, 54.5, 48.6, 36.2, 30.7, 26.3, 25.7, 25.5, 25.4, 23.2, 18.2, 9.9,
9.5. HRMS (ESI): calcd for C29H36ClN4O5, [M-H]− 555.2380, found 555.2382.
3.2.33. (3R,4R,5S)-4-acetamido-5-(3-(4-chlorophenyl)-1-(cyclohexylmethyl)-1H-pyrazole-
5-carboxamido)-3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (16c)
◦
Yield 40%; white solid, mp 232–234 C. 1H NMR (400 MHz, DMSO-d6)
δ 8.36 (d, J = 8.9 Hz,
1H), 8.02 (d, J = 9.1 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 7.15 (s, 1H), 6.64
(s, 1H), 4.37 (ddd, J = 51.7, 13.0, 7.3 Hz, 2H), 4.22–4.07 (m, 2H), 3.88 (dd, J = 19.5, 9.3 Hz,
1H), 3.45–3.30 (m, 1H), 2.56 (dd, J = 17.6, 4.7 Hz, 1H), 2.48–2.37 (m, 1H), 1.85 (s, 1H), 1.73
(s, 3H), 1.61 (d, J = 16.6 Hz, 3H), 1.54–1.29 (m, 7H), 1.18–0.89 (m, 6H), 0.85 (t, J = 7.2 Hz, 3H),
0.78 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.1, 168.0, 159.4, 147.5, 138.0,
137.8, 132.7, 132.0, 130.1, 129.4, 127.0, 104.7, 81.6, 75.6, 56.6, 54.5, 48.6, 39.0, 30.6, 30.4, 26.34,
26.31, 25.7, 25.6, 23.2, 9.9, 9.5. HRMS (ESI): calcd for C31H40ClN4O5, [M-H]− 583.2693,
found 583.2699.
3.2.34. General Procedure for the Synthesis of Compounds 19a–e and 20a–d
To a stirred solution of dimethyl oxalate (1.30 g, 11 mmol) and 1-cyclopropylethan-1-
one (991 µL, 10 mmol) in toluene (60 mL) was added a solution of potassium tert-butoxide
(1.35 g, 12 mmol) in THF (60 mmol). The resulting solution was stirred at room temperature
overnight. The reaction was quenched with 1 N HCl and extracted with ethyl acetate.
The combined organic layers were dried over Na2SO4, filtered, and concentrated under
reduced pressure to provide crude product 18.
Compound 18 was dissolved in EtOH (15 mL) and aryl hydrazine (12 mmol) was
added. The mixture was heated to 85 ◦C for 4 h. The solvent was removed under reduced
pressure and the residue was purified by column chromatography, eluting with a gradient
of hexane/ethyl acetate (15:1 to 8:1) to provide the desired product from 19a
(for 19a–e, the methyl esters were transformed to ethyl esters in EtOH).
–e to 20a–d

Pharmaceuticals 2021, 14, 371
16 of 20
3.2.35. General Procedure for the Synthesis of Compounds 21a–e and 22a–d
The hydrolysis of 19a
for compounds 4a–k.
–e and 20a–d was performed in a similar manner as described
3.2.36. General Procedure for the Synthesis of Compounds 23a–e and 25a–d
The condensation of 21a and 22a with oseltamivir phosphate was performed in a
–
e
–d
similar manner as described for compounds 5a–k.
3.2.37. General Procedure for the Synthesis of Compounds 24a–e and 26a–d
Each compound of 23a
1 M NaOH aqueous solution (2 mL) was added. The mixture was stirred at 50 C overnight.
The solvent was removed in vacuo, the residue was diluted with 10 mL water and washed
–
e
and 25a
–
d
(0.2 mmol) was dissolved in MeOH (10 mL), and
◦
with DCM (10 mL
product which was collected by filtration and dried in vacuo.
×
2). The aqueous phase was acidified by 1 M HCl to precipitate the
3.2.38. (3R,4R,5S)-4-acetamido-5-(5-cyclopropyl-1-phenyl-1H-pyrazole-3-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (24a)
◦
Yield 57%; white solid, mp 273–275 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.96 (d, J = 8.2 Hz,
1H), 7.90 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 7.8 Hz, 2H), 7.57 (t, J = 7.7 Hz, 2H), 7.48 (t, J = 7.2 Hz,
1H), 6.69 (s, 1H), 6.48 (s, 1H), 4.19–4.02 (m, 2H), 3.95 (dd, J = 15.9, 8.6 Hz, 1H), 3.45–3.30
(m, 1H), 2.59 (dd, J = 17.8, 4.5 Hz, 1H), 2.39 (dd, J = 17.8, 7.8 Hz, 1H), 1.83 (td, J = 8.2, 4.2 Hz,
1H), 1.78 (d, J = 10.4 Hz, 3H), 1.55–1.33 (m, 4H), 1.00–0.90 (m, 2H), 0.85–0.73 (m, 8H). 13
C
NMR (100 MHz, DMSO-d6) δ 170.3, 167.9, 161.5, 148.0, 146.7, 139.6, 136.9, 129.9, 129.6, 128.6,
125.2, 103.7, 81.6, 74.6, 52.5, 47.6, 29.8, 26.2, 25.8, 23.1, 9.8, 9.6, 9.2, 9.0, 7.8. HRMS (ESI):
calcd for C27H33N4O5, [M-H]− 493.2456, found 493.2461.
3.2.39. (3R,4R,5S)-4-acetamido-5-(5-cyclopropyl-1-(o-tolyl)-1H-pyrazole-3-carboxamido)-
3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (24b)
◦
Yield 61%; white solid, mp 169–171 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.89 (dd,
J = 8.1, 3.5 Hz, 2H), 7.45 (s, 2H), 7.39 (s, 2H), 6.67 (s, 1H), 6.41 (s, 1H), 4.16–4.04 (m, 2H), 3.92
(dd, J = 16.3, 8.5 Hz, 1H), 3.45–3.30 (M, 1H), 2.58 (dd, J = 17.7, 4.3 Hz, 1H), 2.35 (dd, J = 17.7,
7.8 Hz, 1H), 2.02 (s, 3H), 1.74 (s, 3H), 1.48–1.30 (m, 5H), 0.85–0.80 (m, 2H), 0.80–0.70 (m, 8H).
13C NMR (100 MHz, DMSO-d6)
δ 170.2, 167.8, 161.6, 148.9, 146.5, 138.4, 136.9, 136.1, 131.4,
130.0, 129.9, 128.1, 127.1, 101.5, 81.5, 74.6, 52.6, 47.6, 29.9, 26.1, 25.7, 23.1, 17.3, 9.8, 9.5, 8.8,
8.6, 7.0. HRMS (ESI): calcd for C28H35N4O5, [M-H]− 507.2613, found 507.2617.
3.2.40. (3R,4R,5S)-4-acetamido-5-(5-cyclopropyl-1-(m-tolyl)-1H-pyrazole-3-carboxamido)-
3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (24c)
◦
Yield 53%; white solid, mp 220–222 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.97 (d, J = 8.2 Hz,
1H), 7.90 (d, J = 8.4 Hz, 1H), 7.45 (s, 3H), 7.30 (s, 1H), 6.68 (s, 1H), 6.46 (s, 1H), 4.16–4.06
(m, 2H), 3.95 (dd, J = 15.6, 8.3 Hz, 1H), 3.45–3.40 (m, 1H), 2.58 (dd, J = 17.6, 3.7 Hz, 1H),
2.40 (s, 4H), 1.82 (d, J = 4.6 Hz, 1H), 1.77 (s, 3H), 1.53–1.33 (m, 4H), 0.95 (d, J = 6.9 Hz,
2H), 0.85–0.72 (m, 8H). 13C NMR (100 MHz, DMSO-d6)
δ 170.3, 167.9, 161.5, 147.9, 146.6,
139.6, 139.3, 136.6, 130.0, 129.4, 129.2, 125.7, 122.2, 103.6, 81.7, 74.6, 52.4, 47.4, 29.7, 26.2,
25.8, 23.1, 21.3, 9.8, 9.6, 9.2, 9.0, 7.9. HRMS (ESI): calcd for C28H35N4O5, [M-H]− 507.2613,
found 507.2618.
3.2.41. (3R,4R,5S)-4-acetamido-5-(1-(4-bromophenyl)-5-cyclopropyl-1H-pyrazole-3-
carboxamido)-3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (24d)
◦
Yield 55%; white solid, mp 166–168 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.98 (d, J = 8.2 Hz,
1H), 7.92 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 6.67 (s, 1H), 6.50
(s, 1H), 4.10 (d, J = 5.4 Hz, 2H), 3.98–3.91 (m, 1H), 3.45–3.30 (m, 1H), 2.64–2.54 (m, 1H), 2.38
(dd, J = 17.7, 7.8 Hz, 1H), 1.89–1.80 (m, 1H), 1.76 (s, 3H), 1.52–1.33 (m, 4H), 0.96 (d, J = 7.8 Hz,
2H), 0.85–0.70 (m, 8H). 13C NMR (100 MHz, DMSO-d6)
δ 170.4, 168.0, 161.4, 148.1, 147.0,

Pharmaceuticals 2021, 14, 371
17 of 20
138.9, 136.7, 132.6, 130.1, 127.0, 121.4, 104.3, 81.6, 74.7, 52.6, 47.7, 29.9, 26.2, 25.8, 23.1, 9.8,
9.6, 9.1, 8.9, 7.8. HRMS (ESI): calcd for C27H32BrN4O5, [M-H]− 571.1562, found 571.1562.
3.2.42. (3R,4R,5S)-4-acetamido-5-(5-cyclopropyl-1-(4-(trifluoromethyl) phenyl)
-1H-pyrazole-3-carboxamido)-3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (24e)
◦
Yield 60%; white solid, mp 220–222 C. 1H NMR (400 MHz, DMSO-d6)
δ 8.03 (d, J = 8.2 Hz,
1H), 8.00–7. 90 (m, 5H), 6.69 (s, 1H), 6.56 (s, 1H), 4.11 (d, J = 5.3 Hz, 2H), 3.96 (dd, J = 16.3,
8.5 Hz, 1H), 3.45–3.30 (m, 1H), 2.60 (dd, J = 17.6, 4.2 Hz, 1H), 2.40 (dd, J = 17.7, 7.9 Hz, 1H),
1.99–1.89 (m, 1H), 1.77 (s, 3H), 1.51–1.34 (m, 4H), 1.00 (d, J = 7.6 Hz, 2H), 0.72–0.85 (m, 8H).
13C NMR (100 MHz, DMSO-d6)
δ 170.4, 167.8, 161.2, 148.3, 147.5, 142.8, 137.1, 129.8, 128.5
(d, J = 32.0 Hz), 126.9 (d, J = 3.1 Hz), 126.9 (d, J = 3.9 Hz), 125.3, 124.4 (q, J = 270.6 Hz),
104.9, 81.6, 74.7, 52.7, 47.8, 29.8, 26.2, 25.8, 23.1, 9.8, 9.6, 9.2, 9.0, 8.0. HRMS (ESI): calcd for
C28H32F3N4O5, [M-H]− 561.2330, found 561.2335.
3.2.43. (3R,4R,5S)-4-acetamido-5-(3-cyclopropyl-1-phenyl-1H-pyrazole-5-carboxamido)-3-
(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (26a)
◦
Yield 51%; white solid, mp 267–269 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.96 (d, J = 8.3 Hz,
1H), 7.90 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 7.8 Hz, 2H), 7.57 (t, J = 7.7 Hz, 2H), 7.49 (t, J = 7.3 Hz,
1H), 6.67 (s, 1H), 6.48 (s, 1H), 4.16–4.06 (m, 2H), 3.94 (dd, J = 15.8, 8.7 Hz, 1H), 3.45–3.30
(m, 1H), 2.59 (dd, J = 17.7, 4.7 Hz, 1H), 2.38 (dd, J = 17.8, 7.9 Hz, 1H), 1.84 (td, J = 8.3, 4.1 Hz,
1H), 1.78 (d, J = 11.8 Hz, 3H), 1.43 (qt, J = 13.8, 7.1 Hz, 4H), 1.04–0.89 (m, 2H), 0.86–0.71
(m, 8H). 13C NMR (100 MHz, DMSO-d6)
δ 170.3, 168.0, 161.5, 148.0, 146.7, 139.6, 136.6,
130.1, 129.6, 128.6, 125.2, 103.7, 81.6, 74.7, 52.5, 47.6, 29.8, 26.2, 25.8, 23.2, 9.8, 9.6, 9.2, 9.0, 7.8.
HRMS (ESI): calcd for C27H33N4O5, [M-H]− 493.2456, found 493.2461.
3.2.44. (3R,4R,5S)-4-acetamido-5-(3-cyclopropyl-1-(o-tolyl)-1H-pyrazole-5-carboxamido)-
3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (26b)
◦
Yield 45%; white solid, mp 168–170 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.88 (d, J = 8.1 Hz,
2H), 7.42 (d, J = 26.3 Hz, 4H), 6.67 (s, 1H), 6.41 (s, 1H), 4.20–4.10 (m, 2H), 3.92 (dd, J = 16.2,
8.3 Hz, 1H), 3.45–3.30 (m, 1H), 2.58 (dd, J = 17.6, 3.6 Hz, 1H), 2.36 (dd, J = 17.5, 7.6 Hz, 1H),
2.02 (s, 3H), 1.74 (s, 3H), 1.49–1.31 (m, 5H), 0.90–0.85 (m, 2H), 0.80–0.70 (m, 8H). 13C NMR
(100 MHz, DMSO-d6) δ 170.2, 167.8, 161.6, 148.9, 146.5, 138.4, 137.0, 136.1, 131.4, 130.0, 129.9,
128.1, 127.1, 101.5, 81.5, 74.7, 52.6, 47.6, 29.9, 26.1, 25.7, 23.1, 17.3, 9.8, 9.5, 8.8, 8.6, 7.0. HRMS
(ESI): calcd for C28H35N4O5, [M-H]− 507.2613, found 507.2614.
3.2.45. (3R,4R,5S)-4-acetamido-5-(3-cyclopropyl-1-(m-tolyl)-1H-pyrazole-5-carboxamido)-
3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (26c)
◦
Yield 48%; white solid, mp 159–161 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.97 (d, J = 8.2 Hz,
1H), 7.91 (d, J = 8.4 Hz, 1H), 7.45 (s, 3H), 7.29 (s, 1H), 6.69 (s, 1H), 6.46 (s, 1H), 4.17–4.04
(m, 2H), 3.95 (dd, J = 15.6, 8.2 Hz, 1H), 3.45–3.30 (m, 1H), 2.58 (dd, J = 17.8, 4.0 Hz, 1H),
2.40 (s, 4H), 1.82 (d, J = 4.8 Hz, 1H), 1.77 (s, 3H), 1.50–1.35 (m, 4H), 0.95 (d, J = 7.0 Hz,
2H), 0.85–0.70 (m, 8H). 13C NMR (100 MHz, DMSO-d6)
δ 170.3, 167.9, 161.5, 147.9, 146.6,
139.5, 139.3, 136.8, 129.8, 129.4, 129.2, 125.7, 122.1, 103.6, 81.7, 74.6, 52.4, 47.4, 29.7, 26.2,
25.8, 23.1, 21.3, 9.8, 9.6, 9.2, 9.0, 7.9. HRMS (ESI): calcd for C28H35N4O5, [M-H]− 507.2613,
found 507.2614.
3.2.46. (3R,4R,5S)-4-acetamido-5-(1-(4-bromophenyl)-3-cyclopropyl-1H-pyrazole-5-
carboxamido)-3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylic acid (26d)
◦
Yield 40%; white solid, mp 169–171 C. 1H NMR (400 MHz, DMSO-d6)
δ 7.97 (d, J = 8.0 Hz,
1H), 7.90 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 6.68 (s, 1H),
6.50 (s, 1H), 4.30–4.05 (m, 2H), 3.94 (dd, J = 15.9, 8.2 Hz, 1H), 3.45–3.30 (m, 1H), 2.58
(d, J = 13.8 Hz, 1H), 2.39 (dd, J = 17.3, 7.5 Hz, 1H), 1.91–1.80 (m, 1H), 1.76 (s, 3H), 1.50–1.35
(m, 4H), 0.96 (d, J = 5.9 Hz, 2H), 0.85–0.70 (m, 8H). 13C NMR (100 MHz, DMSO-d6)
δ
170.3,
167.9, 161.3, 148.1, 147.0, 138.9, 136.9, 132.6, 129.9, 127.0, 121.4, 104.3, 81.6, 74.7, 52.6, 47.7,

Pharmaceuticals 2021, 14, 371
18 of 20
29.8, 26.2, 25.8, 23.1, 9.8, 9.6, 9.1, 8.9, 7.8. HRMS (ESI): calcd for C27H32BrN4O5, [M-H]−
571.1562, found 571.1565.
3.3. In Silico Studies
The structure of NA was extracted from PDB 2HU0. Structures of the compounds
and the protein for docking were imported to Maestro 10.2, and the conformations were
generated with the panel “Protein Preparation Wizard” and “LigPrep”, respectively. Molec-
ular docking was performed using the Glide in Maestro 10.2, and ADME properties were
predicted by Qikprop (Maestro 10.2). The docking and ADME calculation processes were
conducted with the default parameters unless otherwise mentioned.
4. Conclusions
The 150-cavity of NA represents a potential binding site for novel NA inhibitors. In
the present study, we designed and synthesized a series of pyrazole-based C-5-NH2-acyl
derivatives of OC. Most of the derivatives exhibited moderate to high inhibitory activity
against NA at the µM level, in which compounds 12c and 26b demonstrated the greatest
activity. Moreover, in silico ADME prediction suggest that the four compounds have
drug-like pharmacokinetic properties. Especially, parameters for oral absorption and cell
permeability indicated the prospect of direct oral administration of the derivatives, such
as 6i, 12c, 24b, and 26b. Further molecular docking studies revealed that the derivatives
could dock into the active site and the 150-cavity as expected. However, calculated binding
energy showed that these derivatives possessed higher binding energy than OC (Table S1),
indicating these modifications would reduce the binding affinity to NA. Considering the
inhibitory activity of compounds 12c and 26b, as well as their docking models, it is expected
that further optimized NA inhibitors could be identified based on the work described in
this study.
C-5-NH2 modification of oseltamivir represents one of the most attractive research
interests in the development of novel NA 150-cavity binders. Previously, we initiated
the strategy of using heterocycles to link oseltamivir and the 150-binding fragments [17].
Inspired by the fact that pyrazole is one of the most widely used heterocycles in drug
discovery, we validated our hypothesis that modifying the oseltamivir C-5-NH2 group
by incorporating the pyrazole moiety has the potential to develop novel NA inhibitors.
This work together with our previous discovery on the development of NA inhibitors
containing other heterocycles will pave a solid basis for the design of novel 150-binders as
NA inhibitors in the future.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/ph14040371/s1, docking models of the products, binding free energy of representative
products, experimental data of the intermediates; 1H and 13C NMR Spectra of the products.
Author Contributions: Conceptualization, X.Y. and C.M.; methodology, X.Y. and C.M.; validation,
J.Y. and L.L.; formal analysis, J.Y. and L.L.; writing—original draft preparation, J.Y.; writing—review
and editing, J.Y., L.L., J.X., P.K.-s.C., X.Y. and C.M.; supervision, X.Y. and C.M.; project administration,
X.Y. and C.M.; funding acquisition, X.Y. and C.M. All authors have read and agreed to the published
version of the manuscript.
Funding: The research was supported by Hong Kong Research Grants Council (PolyU 151000/19M
and C5008-19G (C.M.), CUHK 141079/19M (X.Y.)), Hong Kong Polytechnic University (State Key
Laboratory of Chemical Biology and Drug Discovery, Internal grants 1-ZVPS and G-YBYY, and large
equipment fund), and the Chinese University of Hong Kong (Faculty of Medicine Faculty Innovation
Award No. FIA2018/A/03 (X.Y.)).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are provided within the article and the Supplementary Materials.
Conflicts of Interest: The authors declare no competing financial interest.

Pharmaceuticals 2021, 14, 371
19 of 20
Abbreviations
OC
oseltamivir carboxylate
OP
oseltamivir phosphate
Hemagglutinin
Neuraminidase
HA
NA
NAIs
Neuraminidase inhibitors
ADME absorption, distribution, metabolism and excretion
DCM Dichloromethane
DIPEA N,N-Diisopropylethylamine
HATU 2-(7-Azabenzotriazol-1-yl)-N,N,N ,N -tetramethyluronium hexafluorophosphate
0
0
References
1.
Word Health Organization Report. Available online: https://www.who.int/news/item/13-12-2017-up-to-650-000-people-die-
of-respiratory-diseases-linked-to-seasonal-flu-each-year (accessed on 4 December 2020).
2.
3.
4.
Cox, N.J.; Bender, C.A. The molecular epidemiology of influenza viruses. Semin. Virol. 1995, 6, 359–370. [CrossRef]
Bouvier, N.M.; Palese, P. The biology of influenza viruses. Vaccine 2008, 26 (Suppl. S4), D49–D53. [CrossRef]
Zhuang, Q.; Wang, S.; Liu, S.; Hou, G.; Li, J.; Jiang, W.; Wang, K.; Peng, C.; Liu, D.; Guo, A.; et al. Diversity and distribution of
type A influenza viruses: An updated panorama analysis based on protein sequences. Virol. J. 2019, 16, 85. [CrossRef] [PubMed]
Kilbourne, E.D. Influenza pandemics of the 20th century. Emerg. Infect. Dis. 2006, 12, 9–14. [CrossRef]
Dawood, F.S.; Iuliano, A.D.; Reed, C.; Meltzer, M.I.; Shay, D.K.; Cheng, P.-Y.; Bandaranayake, D.; Breiman, R.F.; Brooks, W.A.;
Buchy, P.; et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus
circulation: A modelling study. Lancet Infect. Dis. 2012, 12, 687–695. [CrossRef]
5.
6.
7.
8.
Jefferson, T.; Demicheli, V.; Rivetti, D.; Jones, M.; Di Pietrantonj, C.; Rivetti, A. Antivirals for influenza in healthy adults:
Systematic review. Lancet 2006, 367, 303–313. [CrossRef]
Noshi, T.; Kitano, M.; Taniguchi, K.; Yamamoto, A.; Omoto, S.; Baba, K.; Hashimoto, T.; Ishida, K.; Kushima, Y.; Hattori, K.; et al.
In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase
PA subunit. Antivir. Res. 2018, 160, 109–117. [CrossRef] [PubMed]
9.
Gubareva, L.V.; Fry, A.M. Baloxavir and Treatment-Emergent Resistance: Public Health Insights and Next Steps. J. Infect. Dis.
2020, 221, 337–339. [CrossRef]
10. Greener, M. Using oseltamivir (Tamiflu) as a first line treatment for seasonal or pandemic flu. Nurs. Times 2009, 105, 22. [PubMed]
11. Gamblin, S.J.; Skehel, J.J. Influenza hemagglutinin and neuraminidase membrane glycoproteins. J. Biol. Chem. 2010, 285,
28403–28409. [CrossRef]
12. Russell, R.J.; Haire, L.F.; Stevens, D.J.; Collins, P.J.; Lin, Y.P.; Blackburn, G.M.; Hay, A.J.; Gamblin, S.J.; Skehel, J.J. The structure of
H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature 2006, 443, 45–49. [CrossRef] [PubMed]
13. Wu, Y.; Qin, G.; Gao, F.; Liu, Y.; Vavricka, C.J.; Qi, J.; Jiang, H.; Yu, K.; Gao, G.F. Induced opening of influenza virus neuraminidase
N2 150-loop suggests an important role in inhibitor binding. Sci. Rep. 2013, 3, 1551. [CrossRef] [PubMed]
14. Xie, Y.; Xu, D.; Huang, B.; Ma, X.; Qi, W.; Shi, F.; Liu, X.; Zhang, Y.; Xu, W. Discovery of N-substituted oseltamivir derivatives as
potent and selective inhibitors of H5N1 influenza neuraminidase. J. Med. Chem. 2014, 57, 8445–8458. [CrossRef]
15. Zhang, J.; Poongavanam, V.; Kang, D.; Bertagnin, C.; Lu, H.; Kong, X.; Ju, H.; Lu, X.; Gao, P.; Tian, Y.; et al. Optimization
of N-Substituted Oseltamivir Derivatives as Potent Inhibitors of Group-1 and -2 Influenza A Neuraminidases, Including a
Drug-Resistant Variant. J. Med. Chem. 2018, 61, 6379–6397. [CrossRef]
16. Zhang, J.; Murugan, N.A.; Tian, Y.; Bertagnin, C.; Fang, Z.; Kang, D.; Kong, X.; Jia, H.; Sun, Z.; Jia, R.; et al. Structure-Based
Optimization of N-Substituted Oseltamivir Derivatives as Potent Anti-Influenza A Virus Agents with Significantly Improved
Potency against Oseltamivir-Resistant N1-H274Y Variant. J. Med. Chem. 2018, 61, 9976–9999. [CrossRef]
17. Ye, J.; Yang, X.; Xu, M.; Chan, P.K.; Ma, C. Novel N-Substituted oseltamivir derivatives as potent influenza neuraminidase
inhibitors: Design, synthesis, biological evaluation, ADME prediction and molecular docking studies. Eur. J. Med. Chem. 2019
182, 111635. [CrossRef] [PubMed]
,
18. Wang, P.; Oladejo, B.O.; Li, C.; Fu, L.; Zhang, S.; Qi, J.; Lv, X.; Li, X. Structure-based design of 50-substituted 1,2,3-triazolylated
oseltamivir derivatives as potent influenza neuraminidase inhibitors. RSC Adv. 2021, 11, 9528–9541. [CrossRef]
19. Wang, K.; Lei, Z.; Zhao, L.; Chen, B.; Yang, F.; Liu, K.; Zhu, H.; Zhao, H.; Cao, R.; Zhang, K.; et al. Design, synthesis and biological
evaluation of oseltamivir derivatives containing pyridyl group as potent inhibitors of neuraminidase for influenza A. Eur. J. Med.
Chem. 2020, 185, 111841. [CrossRef] [PubMed]
20. Wang, K.; Yang, F.; Wang, L.; Liu, K.; Sun, L.; Lin, B.; Hu, Y.; Wang, B.; Cheng, M.; Tian, Y. Synthesis and biological evaluation of
NH2-acyl oseltamivir analogues as potent neuraminidase inhibitors. Eur. J. Med. Chem. 2017, 141, 648–656. [CrossRef] [PubMed]
21. Antilla, J.C.; Baskin, J.M.; Barder, T.E.; Buchwald, S.L. Copper-diamine-catalyzed N-arylation of pyrroles, pyrazoles, indazoles, im-
idazoles, and triazoles. J. Org. Chem. 2004, 69, 5578–5587. [CrossRef]
22. Dawood, K.M.; Abdel-Gawad, H.; Mohamed, H.A.; Badria, F.A. Synthesis, anti-HSV-1, and cytotoxic activities of some new
pyrazole- and isoxazole-based heterocycles. Med. Chem. Res. 2010, 20, 912–919. [CrossRef]

Pharmaceuticals 2021, 14, 371
20 of 20
23. Levent, S.; Caliskan, B.; Ciftci, M.; Ozkan, Y.; Yenicesu, I.; Unver, H.; Banoglu, E. Pyrazole derivatives as inhibitors of arachidonic
acid-induced platelet aggregation. Eur. J. Med. Chem. 2013, 64, 42–53. [CrossRef] [PubMed]
24. Aghazadeh Tabrizi, M.; Baraldi, P.G.; Baraldi, S.; Ruggiero, E.; De Stefano, L.; Rizzolio, F.; Di Cesare Mannelli, L.; Ghelardini, C.;
Chicca, A.; Lapillo, M.; et al. Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent, Reversible, and Selective
Monoacylglycerol Lipase (MAGL) Inhibitors. J. Med. Chem. 2018, 61, 1340–1354. [CrossRef] [PubMed]